PV Loops are the gold-standard method for assessing cardiac function in animal studies. The technique produces load-independent measurements of heart performance, giving the clearest view of cardiac function in typical, disease, and treatment models. It turns out, one of the greatest limits of pressure-volume research is the non-standardized approach to performing and publishing this research.
According to a recent article led by authors Dr. Oliver Wearing and Dr. Chris West (University of British Columbia), lack of clarity in the implementation and reporting of experimental methods has led to difficulties in comparing results between studies. Crucial methodological steps often go unreported. For example, out of a sample of recent publications on this topic only one of 18 essential pieces of information was included in all publications. Each step in the process of pressure-volume catheterization has an influence on the interpretation of the end
Points on the PV Loop.
results, limiting the potential impact of each experiment, complicating our understanding of animal models of interest and fracturing the field of PV research into smaller and smaller subsets.
To address this problem, Drs. Wearing and West set out to bring together a group of leading experts in the preclinical PV field to draw up guidelines that aim to improve reliability and reproducibility. Together, this expert team reviewed existing rodent PV catheterisation research, assessing the variations in methods, equipment, and consumables. The result is a comprehensive list of carefully considered recommendations for standardization that can be applied across disciplines and models. If this attempt to stabilize the field is successful, it would greatly improve the impact of PV research moving forward.
Related: Associate Professor Chris West: The heart, the spine, and the PV loop >>
What should be included in PV publications?
The article lists 18 methodological details from four categories that need to be included in a publication in order to ensure that the experiment/s can be both interpreted and reproduced accurately. This may seem like a lot of information, but specificity is key to success when you're trying to accurately assess and reproduce findings. Without that specificity, you risk comparing data that was collected under wholly different conditions, and potentially drawing incorrect conclusions.
Animal Details/Experimental Setup
- Strain
- Age
- Animal body mass (or indirectly from body surface area)
- Sex
- Anesthetic agent(s), dose, and mode/route of delivery
- Temperature support/control
- Use of mechanical ventilator
- Blood/end-tidal gas monitoring during PV data acquisition
- Open vs. closed-chest approach
PV Catheter
- Catheter technology (i.e., admittance/conductance) or provider
- Volume calibration performed
Data Collection
- Duration of resting PV data collected/analyzed
- Number of occlusions performed/analyzed
- Data acquisition sampling rate
- Type of models used for load-independent variables and 𝝉
Data Reporting
- Heart rate data during PV data acquisition
- Arterial blood pressure during PV data acquisition
- Load-independent indices of systole and/or diastole, and/or 𝝉
Reducing the differences between experiments
Beyond the above methodological information, Dr. Wearing and colleagues also provide recommendations to further align PV research. The better aligned labs are in their experimental designs, the greater the overlap between experiments, and the more cohesive the field becomes as a whole. By improving that overall cohesion, PV researchers will be better able to compare their findings and build a more complete understanding of cardiac function.
Anesthetics
All anesthetics cause some level of cardiovascular disruption, so the choice must be made carefully based on the experimental goals.
- Isoflurane: The most common anesthetic due to its ease of use and mild cardiovascular depressant effects. However, it causes vasodilation and has negative contractility and heart rate effects. Its potent vasodilatory effect in the lungs makes it problematic for right ventricle PV studies. The authors recommend a maintenance dose of <2% for left ventricle studies.
- Barbiturates: These agents, particularly pentobarbital, have well-recognized cardiovascular depressant effects, including direct inhibition of myocardial performance and heart rate.
- Urethane: Provides long-lasting and stable anesthesia with relatively modest cardiovascular depression. It does not cause significant pulmonary vasodilation, making it a good choice for right ventricle studies. However, the route of administration is critical; intravenous delivery is preferred over intraperitoneal injection, which has profound negative metabolic effects. It is also carcinogenic and limited to terminal procedures.
Analgesics and local anesthetics should be used to minimize the required dose of the primary anesthetic agent. The agent, dose, and route of administration should be clearly reported.
Ventilation
Respiration causes swings in intrathoracic pressure that directly affect cardiac filling and can distort PV loops, particularly diastolic parameters and right ventricle measurements.
To standardize these pressure changes, a mechanical ventilator is often preferred, while software can also be employed to sort loops according to certain phases of the ventilatory cycle. If available, a ventilator can produce a brief ‘inspiration hold’ while performing occlusions for load-independent indices of heart function, eliminating any confounding swings in intrathoracic pressure due to ventilation. However, arterial blood gases should be monitored to ensure that any mechanical ventilation is producing physiologically relevant concentrations of dissolved gases in the blood. Whatever method is chosen for a particular study, however, should be clearly reported to properly contextualize the conditions under which the cardiac function was measured.
Related: PV Tips and Tricks: Expert advice for measuring pressure-volume loops in mice >>
Animal Variation
PV indices are subject to variability from factors like animal sex, age, and body mass, electrical noise, and slight differences in catheter placement. Significant variability can also be observed between consecutive occlusions in the same animal.
Recommendations
- Use power analyses to determine appropriate sample sizes (a practice the authors found was absent in all 56 reviewed papers).
- Apply digital filtering or smoothing to the volume signal cautiously and consistently to reduce noise.
- Report data from multiple occlusions to account for within-animal variability.
Volume Calibration
PV catheters measure conductance or admittance, which must be converted to volume units through a calibration process. This requires an independent measure of stroke volume.
Admittance technology can dynamically separate the myocardial contribution to the signal in real-time, whereas conductance technology requires hypertonic saline injection to estimate parallel conductance.
The gold standard for measuring beat-by-beat stroke volume is a perivascular transit-time flow probe placed on the ascending aorta, though this requires an open-chest preparation. Other methods include echocardiography, which is less invasive but may also be less accurate.
Related: Should you be using Admittance or Conductance calibration for PV Loops? >>
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Determining Load-Independent Indices
To assess load-independent function, the heart's loading conditions must be rapidly manipulated by occluding the inferior vena cava to reduce preload and, if necessary, the abdominal aorta to increase afterload.
Occlusions should be brief (10–15 seconds) to avoid activating confounding autonomic reflexes. For systolic indices, three replicate inferior vena cava occlusions should be performed, with hemodynamics allowed to return to baseline between each. A single, brief abdominal aorta occlusion can follow if a full assessment of diastolic function is needed.
Schematic of preload reductions using an IVC occlusion to calculate the EDPVR, Ees, and EDPVR.
Key Systolic Indices to Report:
- Preload-recruitable stroke work: Relates myocardial work to preload. It is robustly linear and less influenced by chamber size than end-systolic elastance.
- dP/dtmax-EDV: An index of early systolic performance indexed to preload.
- End-systolic elastance: A measure of chamber stiffness. Its underlying relationship (the ESPVR) is often curvilinear, so Wearing et al. recommend using a nonlinear regression model for analysis.
Schematic of ESPVR and changes in cardiac contractility.
Key Diastolic Indices to Report:
- End-diastolic pressure-volume relationship (EDPVR): An index of passive ventricular stiffness. Its characterization is improved by performing both inferior vena cava and abdominal aorta occlusions.
- Tau (τ): A time constant measuring active (isovolumic) relaxation. Drs. Wearing and West, and their expert co-authors, recommend the logistic model (τLogistic) as it is the most robust and load-independent method of calculation.
Schematic depicting changes in left ventricular compliance as a result of changes in the slope of the EDPVR.
Acquiring and Reporting PV Data
Researchers often reduce thousands of heartbeats of baseline data into a single average value, losing valuable information about beat-to-beat variability.
Recommendations
- Use a minimum data sampling rate of 2 kHz to preserve the integrity of the signals, especially for diastolic parameters.
- Instead of reporting only a mean value, present data with measures of variance (e.g., standard deviation, range).
- Visualize the distribution of the data by presenting frequency histograms for indices of interest.
Related: Introduction to PV loops: Understanding points on the PV loop and measures of cardiac function >>
Related: Pressure-Volume (PV) Loop Analysis Software >>
Find out more about the guidelines here:
Guidelines for assessing ventricular pressure-volume relationships in rodents >>
